INFECTION AND IMMUNITY, Apr. 1999, p. 1729–1735 Vol. 67, No.

4
0019-9567/99/$04.0010
Copyright © 1999, American Society for Microbiology. All Rights Reserved.

A Novel 62-Kilodalton Egg Antigen from Schistosoma mansoni

Induces a Potent CD41 T Helper Cell Response in the
C57BL/6 Mouse
HIROKO ASAHI, HECTOR J. HERNANDEZ, AND MIGUEL J. STADECKER*
Department of Pathology, Tufts University School of Medicine, Boston, Massachusetts 02111
Received 6 November 1998/Returned for modification 16 December 1998/Accepted 14 January 1999

In infection with Schistosoma mansoni, hepatic granuloma formation is mediated by CD41 T helper (Th) cells
sensitized to schistosomal egg antigens. There is considerable variation among infected individuals with
respect to both severity of disease and the T-cell response to egg antigens. In the BL/6 mouse, the egg
granulomas are relatively small and the relevant sensitizing egg antigens are largely unknown. We investigated
the CD41 Th cell response of infected BL/6 mice to egg antigens fractionated by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis and found a prominent lymphoproliferative response to be directed against
a 62-kDa component. With the aid of a specific T-cell hybridoma, 4E6, the 62-kDa antigen was isolated;
following partial digestion with endoproteinase Glu-C, an internal amino acid sequence was found to be
identical with one present in the enzyme phosphoenolpyruvate carboxykinase (PEPCK) of the organisms
Caenorhabditis elegans and Treponema pallidum and to differ by one residue from PEPCK of various other
species. In CD41 Th cells from 7.5- 8.5-week-infected BL/6 mice, the purified 62-kDa molecule elicited a potent
proliferative response which, based on cytokine analysis, was of a mixed Th-1 and Th-2 type. Our results reveal
a novel egg antigen of particular prominence in the BL/6 mouse and suggest that the immune response in
schistosomiasis is a product of sensitization to egg antigens that may vary considerably in immunogenicity
from strain to strain.

The immunopathological damage in schistosomiasis man-
soni consists mainly of granulomatous inflammation around
parasite eggs in the liver and intestines, which may lead to
scarring, portal hypertension, hemorrhage, and death (2, 34).
There is considerable variation in the overall severity of this
disease, both in humans and in mice. For example, mice of the
C3H and CBA strains develop significantly larger egg granu-
lomas than do those of the BL/6 strain (5, 9).
Granulomatous inflammation is a complex cellular hyper-
sensitivity reaction that involves recruitment and activation of
several types of inflammatory cells, the interplay of numerous
mediators, including cytokines, and the synthesis of matrix
proteins. Granuloma formation is now known to be strictly
dependent on CD41 T helper (Th) cells specific for schistoso-
mal egg antigens (SEA) (14, 23), and by virtue of signature
cytokines, there is strong evidence that it can be mediated by
Th-1 and Th-2-type CD41 Th cells (7, 13, 28, 36, 46). The
CD41 Th cells become activated following specific recognition
of accessory cell-bound major histocompatibility complex
(MHC) class II molecules harboring selected SEA-derived
peptides. However, identification of individual T-cell-sensitiz-
ing egg antigens and derived peptides is only at a very early
stage.
We previously investigated the nature of the anti-SEA T-cell
repertoire by using CD41 Th cells from infected mice as well
as panels of specific T-cell hybridomas. We found that C3H
mice display strong polyclonal T-cell responses against the
major egg antigen Sm-p40, whereas in BL/6 mice the response
is much weaker (15). Moreover, the specificity analysis of the
derived T-cell hybridomas suggested that in C3H mice, a sig-
* Corresponding author. Mailing address: Department of Pathology,
Tufts University School of Medicine, 136 Harrison Ave., Boston, MA
02111. Phone: (617) 636-6732. Fax: (617) 636-2990. E-mail: mstadeck
@opal.tufts.edu.
nificant proportion of the anti-SEA T-cell repertoire is di-
rected against Sm-p40, whereas no hybridomas derived from
BL/6 mice recognized this antigen (15). These findings imply
that the immunopathology in the BL/6 strain is largely directed
against antigens other than Sm-p40. They also raise the possi-
bility that differences in egg antigen recognition patterns in
mouse strains of different haplotypes may be reflected in the
overall magnitude of the resulting immunopathology.
To gain insight into the basis of the immune response to
schistosome eggs in BL/6 mice, we used specific T-cell hybrid-
omas as monoclonal probes to discern which SEA components
they recognize. We now report on the identification and char-
acterization of a novel 62-kDa egg antigen, found to elicit a
particularly strong response in CD41 Th cells from infected
mice of the BL/6 strain.
MATERIALS AND METHODS
Infection of mice. Six- to eight-week-old female BL/6 (H-2b) and CBA (H-2k)
mice were purchased from the Jackson Laboratory (Bar Harbor, Maine). Some
of the mice were infected intraperitoneally with 70 cercariae of Schistosoma
mansoni (Puerto Rico strain). Cercariae were shed from infected Biomphalaria
glabrata snails, provided to us by the Biomedical Research Institute, Rockville,
Md.
Antigens. SEA was prepared as described previously (1) at the Center for
Tropical Diseases, University of Massachusetts at Lowell, and was obtained in
part through the UNDP/World Bank/WHO Special Programme for Research
and Training in Tropical Diseases. Recombinant antigen Sm-p40 (amino acids 94
to 341) was prepared as previously described (15). Protein concentrations were
determined by a modification of the Bradford method using the Coomassie Plus
protein assay reagent (Pierce, Rockford, Ill.).
CD41 Th cell responses. CD41 Th cells were isolated from mesenteric lymph
node cells of mice after 7.5 to 8.5 weeks of infection. The cells were purified by
negative selection as described elsewhere (15). The procedure involves nylon
wool filtration and two cycles of incubation with monoclonal antibodies (MAbs)
against I-Ab/I-Ek, heat-stable antigen and CD8, and complement. To assess
proliferation, 1.5 3 105 CD41 Th cells plus 4 3 105 syngeneic irradiated (with
3,000 rads) normal splenocytes were cultured in a volume of 200 ml for a total of
114 h in the presence of the indicated antigens. During the last 18 h of culture,
0.5 mCi of tritiated thymidine ([3H]dThd) was added to each well, and incorpo-

1729
1730 ASAHI ET AL. INFECT. IMMUN.

ration into DNA was measured by liquid scintillation spectroscopy; data pre-
sented represent means 6 standard deviations (SD).
T-cell hybridoma 4E6. The SEA specific T-cell hybridoma 4E6 was derived
from BL/6 mice as described elsewhere (15). Antigen reactivity of this hybridoma
was determined as described previously (15), with some modifications. Briefly,
106 4E6 hybridoma cells, together with 3 3 106 irradiated (with 3,000 rads)
normal syngeneic splenocytes, were cultured either with graded concentrations
of soluble proteins for 24 h or with proteins immobilized on nitrocellulose
membranes (NC; Immobilon NC Pure; Millipore Corporation, Bedford, Mass.)
for 42 h. Positive hybridoma responses were assessed by incubation of the culture
supernatants with 9 3 103 HT-2 indicator cells for 22 h (44).
Determination of antigen-specific cytokine production. CD41 Th cells (106)
plus 4 3 106 irradiated splenic antigen-presenting cells were cultured in 1-ml
volumes together with graded concentrations of the indicated antigens in 48-well
plastic plates. The cytokines gamma interferon (IFN)-g, interleukin-2 (IL-2),
IL-4, and IL-5 were measured in culture supernatants by enzyme-linked immu-
nosorbent assay (ELISA), with corresponding cytokine-specific capture MAbs,
detection MAbs, standard cytokines, and protocols from PharMingen (San Di-
ego, Calif.). The cytokines were measured after optimal culture times, which
were 24 h for IL-2 and 48 h for IFN-g, IL-4, and IL-5.
SDS-PAGE and preparation of immunoblots. Test materials were boiled in
reducing sample buffer containing 2% sodium dodecyl sulfate (SDS) and 1%
2-mercaptoethanol (2-ME) and then separated by reduced sodium dodecyl sul-
fate-polyacrylamide gel electrophoresis (SDS-PAGE) (20). For preparation of
immunoblots, 85 mg of protein of SEA was loaded on each lane. Proteins were
transferred from gels to NC by electroblotting with 25 mM Tris–192 mM glycine
buffer containing 10% methanol (pH 8.4) (41). The procedure was carried out
overnight at a constant voltage of 15 V in a tank transfer system (Bio-Rad
Laboratories, Hercules, Calif.). One lane of the blot was cut off, stained with
amido black (0.1% in 5% acetic acid), and used as a reference to localize
separated proteins on unstained NC. Each NC blot was cut into 30 sections
corresponding to different molecular masses. Each section was cut further into
smaller parts, sterilized by exposure to 15,000 rads of g irradiation, and tested for
the ability to stimulate CD41 Th cell populations and the T-cell hybridoma.
Corresponding pooled sections from two lanes were used for each culture well of
hybridoma cells, and those from one half lane were used for the CD41 Th cells.
For preparation of controls strips, SEA (50 mg of protein) or phosphate-buffered
saline (50 ml) was directly applied to 60 mm2 of NC.
Electroelution. Protein fractions separated by SDS-PAGE were stained with a
reversible copper stain (Bio-Rad Laboratories), excised from the gels, destained,
and eluted for more than 24 h in an electroeluter (Bio-Rad Laboratories), using
a volatile elution buffer containing 50 mM NH4HCO3, 0.1% SDS, and 0.1%
2-ME. Subsequent to elution, the SDS was removed by overnight electrodialysis
in the same tank following replacement with fresh buffer made without SDS and
2-ME. Purity, molecular weight, and concentration of proteins isolated from gels FIG. 1. Representative SDS-PAGE (7.5% gel) profile of SEA on immuno-
were assessed on silver-stained gels with an image analyzing system (Molecular blots stained with amido black. Molecular weight marker standards are indicated
Analyst 2.1; Bio-Rad Laboratories). on the left. The prominent band of fraction 15 represents the Sm-p40 antigen.
Limited proteolytic digestion. Materials isolated from reduced SDS-polyacryl- Frs., fractions.
amide gels were electroeluted and digested with 20 mg endoproteinase Glu-C
(Staphylococcus aureus V8 protease; Promega, Madison, Wis.) per ml in the
presence of 50 mM Tris-HCl buffer (pH 6.8), 20% glycerin, 0.01% bromophenol
blue dye, 0.1% SDS, and 10 mM EDTA, essentially as described previously (18). sentative experiment depicting the various molecules with their
Protein sequencing. After digestion with V8 protease, the proteolytic frag-
ments were separated by SDS-PAGE as described above except that test mate- apparent molecular masses are shown in Fig. 1. Individual
rials were not reduced or boiled. For the SDS-PAGE, 0.1 mM sodium thiogly- components were subsequently excised from the immunoblots
colate was added to the electrophoresis buffer in the upper chamber. and tested for the ability to stimulate CD41 Th cells from
Electrophoresis of V8 protease digests (10% gel) was typically run at 50 V for infected BL/6 mice. Results from a representative experiment
stacker gels and 120 V for separating gels at constant voltage. Fractions were
electroeluted as described above in buffer without 2-ME and tested for the ability shown in Fig. 2A indicate that the CD41 Th cells reacted
to stimulate hybridoma cells. After separation, V8 protease digests were also against several fractionated SEA components; however, the
electroblotted to a polyvinylidene difluoride membrane (Immobilon-PSQ; Milli- response to fraction 7 was among the strongest.
pore) (24). The electroblot transfer was carried out for 70 min at 4°C at 50 V, T-cell hybridoma 4E6 recognizes a strongly stimulatory SEA
using blotting buffer consisting of 25 mM Tris, 192 mM glycine, and 10% meth-
anol. The blots were stained with 0.1% Coomassie blue G in 50% methanol and component. In addition to the CD41 Th cells, a panel of
destained with 10% acetic acid in 50% methanol. After being washed with water, SEA-specific BL/6 T-cell hybridomas was screened with the
the blots were dried and the stimulatory fractions were excised for protein fractionated SEA components. One of these T-cell hybrid-
sequencing, which was carried out on an Applied Biosystems gas-phase se- omas, 4E6, responded to a potent antigen contained in fraction
quencer (model 477A) at the Tufts Core Facility, Department of Physiology,
Tufts University School of Medicine. The sequence was compared with se- 7 (Fig. 2B). Following excision of the relevant area and elec-
quences from known proteins with BLAST (Basic Local Alignment Search Tool) troelution from gels, the stimulatory component appeared as a
programs from the National Center for Biotechnology Information. single band on silver-stained gels, with an apparent molecular
mass of 62 kDa (Fig. 3A). The identified 62-kDa antigen elic-
RESULTS ited a dose-dependent stimulation of hybridoma 4E6, as de-
termined by the proliferation of HT-2 indicator cells; a protein
Identification of SEA components that stimulate BL/6 concentration of 100 ng/ml was sufficient to elicit a significant
CD41 Th cells. An initial experiment was conducted to exam- response (Fig. 3B).
ine the relative immunogenicity of the various egg antigens in Partial amino acid sequence of the 62-kDa antigen. When
schistosome-infected BL/6 mice. For this purpose, SEA was initially tested for the purpose of amino acid sequencing, the
fractionated by SDS-PAGE; this procedure resulted in the 62-kDa antigen was found to have a blocked amino terminus.
separation of 30 discernible components. Results of a repre- For this reason it was subjected to limited proteolytic digestion
VOL. 67, 1999 IDENTIFICATION OF A NOVEL SCHISTOSOMAL EGG ANTIGEN 1731

FIG. 2. CD41 Th cell responses (A) and 4E6 T-cell hybridoma responses (B) to SDS-PAGE fractions on immunoblots of SEA. CD41 Th cells were isolated from
mesenteric lymph nodes of 8.5-week-infected BL/6 mice. Cell culture conditions in each case were as described in Materials and Methods. [3H]dThd incorporation was
assessed by liquid scintillation spectroscopy. Data are expressed as means 6 1 SD. Background radioactivity from cultures in the absence of antigen is subtracted. Panels
on the right reflect corresponding CD41 Th cell or hybridoma stimulation in the presence of NC, NC coated with 50 mg of SEA (SEA-NC), or the indicated amounts
of SEA. Experiments with CD41 Th cells as well as with the T-cell hybridoma were repeated twice, with similar results. Frs., fractions.

with V8 protease, which cleaves peptides at the carboxyl end of
Glu residues (45). This treatment yielded several different
fragments. Discernible fragments were electroeluted and used
to stimulate hybridoma 4E6; the fragments were also reexam-
ined for purity on silver-stained SDS-polyacrylamide gels un-
der nonreducing conditions. Figure 4A shows 62-kDa fragment
g, which displayed stimulatory activity 55 to 71% greater than
those of the intact and digested molecules; Fig. 4B shows the
migration on SDS-PAGE of fragment g, which has an apparent
molecular mass of 26 to 27 kDa. The V8 protease of 28 kDa
displayed an electrophoretic mobility similar to that of frag-
ment g but, as expected, had no stimulatory activity (not
shown).
Identification of the 62-kDa antigen. Because of its strong
stimulatory activity, fragment g (Fig. 4B) was subjected to
amino acid sequencing. This procedure yielded a 10-amino-
acid peptide with the sequence AGFFGVAPGT (Fig. 5). Com-
puter search of GenBank disclosed that this sequence, together
with a deduced Glu residue (the site of V8 protease cleavage),
is identical to one found in phosphoenolpyruvate carboxyki-
nase (PEPCK-GTP; EC 4.1.1.32). PEPCK is an enzyme of the
gluconeogenic pathway and has been described for a variety of
species, including helminths, bacteria, fungi, vertebrates, and
arthropods. In the species listed in Fig. 5, PEPCK is composed
of 608 to 651 amino acids. The sequence obtained from our
62-kDa antigen is typically located in the midportion of the
molecule and is completely identical to that in PEPCK of the
organisms Caenorhabditis elegans and Treponema pallidum;
moreover, it varies by only one residue (Ala replaced with
either Ser, Asn, Arg, Tyr, or Phe) with respect to PEPCK of
other species listed in Fig. 5. A secondary sequence was also
found in proteolytic fragment g; this corresponded to the V8
protease.
Polyclonal CD41 Th cell responses to the 62-kDa antigen.
To ascertain its relative immunogenicity, the 62-kDa antigen
was used to stimulate proliferative and cytokine responses in
polyclonal CD41 Th cells from infected mice. Figure 6A shows
that in BL/6 mice, low concentrations of the 62-kDa compo-
nent elicited a potent dose-dependent proliferative response
compared to unfractionated SEA; this response was vastly
stronger than that induced by Sm-p40, which is a demonstrated
major egg immunogen in mice of the H-2k haplotype (4, 15,
16). By comparison, the 62-kDa component also induced a
significant proliferative response in the CBA (H-2k) mouse,
1732 ASAHI ET AL.
FIG. 3. Identification of a 62-kDa antigen recognized by T-cell hybridoma
4E6. (A) Eluted SDS-PAGE gel fraction 7 (Fr.7) (Fig. 1) from 20 mg of SEA,
examined for purity on a 10% silver-stained SDS-polyacrylamide gel, shown next
to total SEA and molecular weight marker standards. (B) Dose response of
T-cell hybridoma 4E6 to 62-kDa antigen as measured by HT-2 indicator cell
proliferation. Data are expressed as mean 6 1 SD. Background radioactivity
from hybridoma cultures in the absence of antigen is subtracted.
although in this case it was considerably smaller than that
elicited by the Sm-p40 antigen (Fig. 6B).
Examination by ELISA of culture supernatants from CD41
Th cells obtained from 8.5-week-infected BL/6 mice revealed
that stimulation with the 62-kDa antigen elicited significant
secretion of IFN-g, IL-4, and IL-5 but smaller amounts of IL-2
(Fig. 7). Interestingly, the 62-kDa antigen by itself stimulated
more IFN-g than unfractionated SEA; however, production of
IL-2, IL-4, and IL-5 was substantially lower than that elicited
by unfractionated SEA. Moreover, except for IFN-g, there was
virtually no cytokine production in response to Sm-p40. In
CBA mice, the 62-kDa antigen elicited relatively little cytokine
secretion in comparison with total SEA, even though, except
for IL-4, overall cytokine production in response to SEA was
markedly higher than in the BL/6 strain. Another striking at-
tribute of the CBA strain is the elevated IFN-g and IL-2
response to Sm-p40, which contrasted sharply with little IL-5
production and virtually no IL-4.
DISCUSSION
Hepatointestinal granuloma formation and the ensuing po-
tentially lethal pathologic events in schistosomiasis mansoni
are strictly dependent on, and mediated by, CD41 Th cells
specific for egg antigens. Although previous reports described
several T-cell-stimulatory activities (3, 12, 21), the precise iden-
tity of most sensitizing egg antigens remains largely unknown.
Given the potential of modifying the immunopathology in ex-
perimental schistosomiasis by manipulating the underlying egg
antigen-specific CD41 Th cell response, our laboratory has
developed monoclonal T-cell hybridomas from sensitized mice
as probes to identify individual egg components. The reasoning
behind this approach is the likelihood that the majority of such
clones will have specificities for the most immunogenic of the
INFECT. IMMUN.
FIG. 4. Stimulation of the 4E6 T-cell hybridoma with V8 protease fragments
of the 62-kDa antigen. (A) Stimulation of T-cell hybridoma 4E6 by, from left to
right, the intact 62-kDa antigen, the fragments of the 62-kDa antigen derived
from digestion with 2 mg of V8 protease, and the most stimulatory fragment g.
Each of these stimulatory activities was obtained from an initial 450 mg of SEA.
Data are expressed as mean 6 1 SD. Background radioactivity from hybridoma
cultures in the absence of antigen is subtracted. (B) Corresponding silver-stained
SDS-PAGE profile of, from left to right, the 62-kDa antigen, the combined V8
protease fragments of the 62-kDa antigen, and fragment g. Five percent of each
antigen preparation used for stimulation was examined on the SDS-polyacryl-
amide gel. Part of the V8 protease migrated close to fragment g but had no
stimulatory activity (not shown). The position of intact V8 protease (28 kDa) is
shown.
egg components, which then could be tracked down and iden-
tified.
This paper reports on such an event in which the highly
sensitive T-cell hybridoma 4E6 was instrumental in the iden-
tification of a strongly stimulatory 62-kDa egg component in
the BL/6 mouse. Limited proteolytic digestion of this compo-
nent yielded stimulatory fragment g which, in turn, facilitated
internal amino acid sequencing of a peptide identical or similar
to one contained in PEPCK of various species. It is likely that
hybridoma 4E6 recognizes the same epitope in the intact 62-
kDa molecule as in fragment g and that the difference in
stimulation is due to a concentration effect. Although not pre-
viously recognized as an egg antigen, PEPCK has been iden-
tified in adult worms and sporocysts of S. mansoni (39, 40).
Moreover, a cDNA clone from a genomic library of S. mansoni
has been partially sequenced (bases 1 to 304) and shown to be
homologous with PEPCK (29).
In contrast to an impressive number of cloned S. mansoni
worm antigens (11, 27, 30, 31, 35, 38), the list of identified S.
mansoni antigens preferentially expressed in eggs is still in its
beginning stage, although Fig. 1 and 2A suggest several poten-
tial immunogenic egg components possibly representing pre-
viously reported candidate antigens (17, 25, 37). Of these,
undoubtedly the most abundant component (fraction 15 in Fig.
1) has been identified as Sm-p40, which is a small heat shock
VOL. 67, 1999 IDENTIFICATION OF A NOVEL SCHISTOSOMAL EGG ANTIGEN 1733

been found to be dominant in the C3H and CBA strains (6,

FIG. 5. Internal amino acid sequence obtained from fragment g and related
sequences in other species corresponding to PEPCK. The 10-amino-acid se-
quence is shown at the top. Vertical lines indicate identical residues; points
indicate mismatched residues. The location (p) of the 10-mer together with the
deduced Glu (E) residue (the site of V8 protease cleavage) within PEPCK is
indicated for each species. The total number of amino acids (aa) in each PEPCK
is indicated in parentheses.
protein with homologies to alpha-crystallins (26). Sm-p40 is a
potent egg immunogen which elicits a strong Th-1-type re-
sponse in C3H and CBA mice, which develop large egg gran-
ulomas (4, 15, 16). Sm-p40 has been fully sequenced (26) and
found to have at least three T-cell epitopes, of which one has
16).
Sm-p40 and the novel 62-kDa antigen lend themselves to
interesting and important comparisons. A strong T-cell re-
sponse to Sm-p40 is restricted by H-2k, and only a minor
reactivity is detected in BL/6 (H-2b) mice as well as mice of the
H-2d and H-2q haplotypes (16). In contrast, the 62-kDa antigen
elicits the major T-cell response in BL/6 mice, although CBA
mice do react to it albeit with a response significantly weaker
than that directed against Sm-p40. Another contrast is that
Sm-p40 preferentially stimulates Th-1-type cytokine produc-
tion, whereas the cytokines elicited in the BL/6 strain by the
62-kDa antigen appear to be of a mixed Th-1 and Th-2 type. Of
interest, however, is that while in BL/6 mice the overall cyto-
kine response is markedly lower, the 62-kDa antigen elicits a
stronger IFN-g response by itself than when included in SEA,
surprisingly at a time of infection (8.5 weeks) when the anti-
SEA cytokine response in this strain is known to shift to the
Th-2 type (28, 36).
Even though the reason for the difference in severity of
schistosomal infection among individual humans and different
mouse strains is not known, there is strong evidence of an
underlying genetic control. In humans, a more pronounced
disease course has been associated with genes within (19, 33,
43) or outside (22) the MHC complex. In mice, both kinds of
genes have been similarly implicated (5). Non-MHC genes
could influence the severity of infection by controlling a variety
of factors involved in the inflammatory and repair processes,
including cytokines, costimulatory and adhesion molecules,
and extracellular matrix components. On the other hand,
MHC control could be exerted by determining the number of
epitopes, if any, that are recognized in each antigen and dic-
tating the type of ensuing T-cell response, as suggested herein.
For example, it has been shown that a Plasmodium falciparum
circumsporozoite epitope is restricted by I-Ab (8, 10); in con-

FIG. 6. Proliferative responses of CD41 Th cells from BL/6 (A) and CBA (B) mice to the 62-kDa antigen. CD41 Th cells were isolated from mesenteric lymph
nodes of 8.5-week-infected mice. Culture conditions were as described in Materials and Methods. [3H]dThd incorporation was assessed by liquid scintillation
spectroscopy. Data are expressed as mean 6 1 SD. Also shown for comparison are responses to Sm-p40 and SEA. Background radioactivity from cultures in the absence
of antigen is subtracted. The same pattern of stimulation was observed when cells from 7.5-week-infected mice were used (not shown).
1734 ASAHI ET AL.
FIG. 7. Cytokine production by CD41 Th cells from BL/6 and CBA mice stimulated with the 62-kDa antigen. CD41 Th cells were isolated from mesenteric lymph
nodes of 8.5-week-infected mice. Culture conditions were as described in Materials and Methods. The cytokines IFN-g, IL-2, IL-4, and IL-5 were measured in culture
supernatants by ELISA. Data are expressed as mean 6 1 SD. Also shown for comparison are responses to Sm-p40 and SEA. The same pattern of cytokine production
was observed when cells from 8-week-infected mice were used (not shown).
trast, BL/6 mice do not respond to an 86-kDa antigen from
adult schistosome worms (32), nor do they make immunoglob-
ulin G antibodies to the Sm-p40 antigen (42).
An important question that remains to be answered is to
what extent the genetically determined recognition and re-
sponse to an antigen influences the outcome of immunopa-
thology and clinical disease. Our findings suggest that the rel-
ative immunogenicity of different schistosomal egg antigens
varies from strain to strain and, extrapolating to outbred pop-
ulations, possibly from individual to individual. However, the
identification of egg antigens potentially has important practi-
cal implications because the most pathogenesis-related re-
sponses could be targeted for specific down-regulation.
ACKNOWLEDGMENTS
This work was supported in part by Public Health Service grant AI
18919 and by the UNDP/World Bank WHO Special Program for
Research and Training in Tropical Diseases. B. glabrata cercariae were
provided by the Biomedical Research Institute, Rockville, Md., under
NIH-NIAID contract N01 AI-55260.
We thank David Stollar and Peter Brodeur for critically reading the
manuscript and Philip LoVerde for helpful discussions.
REFERENCES
1. Boros, D. L., and K. S. Warren. 1970. Delayed hypersensitivity-type granu-
loma formation and dermal reaction induced and elicited by a soluble factor
INFECT. IMMUN.
isolated from Schistosoma mansoni eggs. J. Exp. Med. 132:488–507.
2. Boros, D. L. 1989. Immunopathology of Schistosoma mansoni infection. Clin.
Microbiol. Rev. 2:250–269.
3. Brown, A. P., H. G. Remold, K. S. Warren, and J. R. David. 1977. Partial
purification of antigens from eggs of Schistosoma mansoni that elicit delayed
hypersensitivity. J. Immunol. 119:1275–1278.
4. Cai, Y., J. G. Langley, D. I. Smith, and D. L. Boros. 1996. A cloned major
Schistosoma mansoni egg antigen with homologies to small heat shock pro-
teins elicits Th1 responsiveness. Infect. Immun. 64:1750–1755.
5. Cheever, A. W., R. H. Duvall, T. A. Hallack, Jr., R. G. Minker, J. D. Malley,
and K. G. Malley. 1987. Variation of hepatic fibrosis and granuloma size
among mouse strains infected with Schistosoma mansoni. Am. J. Trop. Med.
Hyg. 37:85–97.
6. Chen, Y., and D. L. Boros. 1998. Identification of the immunodominant T
cell epitope of p38, a major egg antigen, and characterization of the epitope-
specific Th responsiveness during murine schistosomiasis mansoni. J. Immu-
nol. 160:5420–5427.
7. Chikunguwo, S. M., T. Kanazawa, Y. Dayal, and M. J. Stadecker. 1991. The
cell-mediated response to schistosomal antigens at the clonal level. In vivo
functions of cloned murine egg antigen-specific CD41 T helper type 1 lym-
phocytes. J. Immunol. 147:3921–3925.
8. Del Giudice, G., J. A. Cooper, J. Merino, A. S. Verdini, A. Pessi, A. R. Togna,
H. D. Engers, G. Corradin, and P. H. Lambert. 1986. The antibody response
in mice to carrier-free synthetic polymers of Plasmodium falciparum circum-
sporozoite repetitive epitope is I-Ab-restricted: possible implications for
malaria vaccines. J. Immunol. 137:2952–2955.
9. Fanning, M. M., P. A. Peters, R. S. Davis, J. W. Kazura, and A. A. Mahmoud.
1981. Immunopathology of murine infection with Schistosoma mansoni: re-
lationship of genetic background to hepatosplenic disease and modulation.
J. Infect. Dis. 144:148–153.
10. Good, M. F., J. A. Berzofsky, W. L. Maloy, Y. Hayashi, N. Fujii, W. T.
Hockmeyer, and L. H. Miller. 1986. Genetic control of the immune response
VOL. 67, 1999 IDENTIFICATION OF A NOVEL SCHISTOSOMAL EGG ANTIGEN
in mice to a Plasmodium falciparum sporozoite vaccine. Widespread nonre-
sponsiveness to single malaria T epitope in highly repetitive vaccine. J. Exp.
Med. 164:655–660.
11. Grezel, D., M. Capron, J. M. Grzych, J. Fontaine, J. P. Lecocq, and A.
Capron. 1993. Protective immunity induced in rat schistosomiasis by a single
dose of the Sm28GST recombinant antigen: effector mechanisms involving
IgE and IgA antibodies. Eur. J. Immunol. 23:454–460.
12. Harn, D. A., K. Danko, J. J. Quinn, and M. J. Stadecker. 1989. Schistosoma
mansoni: the host immune response to egg antigens. I. Partial characteriza-
tion of cellular and humoral responses to pI fractions of soluble egg antigens.
J. Immunol. 142:2061–2066.
13. Henderson, G. S., X. Lu, T. L. McCurley, and D. G. Colley. 1992. In vivo
molecular analysis of lymphokines involved in the murine immune response
during Schistosoma mansoni infection. II. Quantification of IL-4 mRNA,
IFN-gamma mRNA, and IL-2 mRNA levels in the granulomatous livers,
mesenteric lymph nodes, and spleens during the course of modulation. J. Im-
munol. 148:2261–2269.
14. Hernandez, H. J., Y. Wang, N. Tzellas, and M. J. Stadecker. 1997. Expres-
sion of class II, but not class I, major histocompatibility complex molecules
is required for granuloma formation in infection with Schistosoma mansoni.
Eur. J. Immunol. 27:1170–1176.
15. Hernandez, H. J., W. C. Trzyna, J. S. Cordingley, P. H. Brodeur, and M. J.
Stadecker. 1997. Differential antigen recognition by T cell populations from
strains of mice developing polar forms of granulomatous inflammation in
response to eggs of Schistosoma mansoni. Eur. J. Immunol. 27:666–670.
16. Hernandez, H. J., C. M. Edson, D. A. Harn, C. J. Ianelli, and M. J. Sta-
decker. 1998. Schistosoma mansoni: genetic restriction and cytokine profile
of the CD41 T helper cell response to dominant epitope peptide of major
egg antigen Sm-p40. Exp. Parasitol. 90:122–130.
17. Hirsch, C., C. A. Almeida, B. L. Doughty, and A. M. Goes. 1997. Character-
ization of Schistosoma mansoni 44.7/56.8 kDa egg antigens recognized by
human monoclonal antibodies which induce protection against experimental
infection and proliferation of peripheral blood mononuclear cells from schis-
tosomiasis patients. Vaccine 15:948–954.
18. Kennedy, T. E., M. A. Gawinowicz, A. Barzilai, E. R. Kandel, and J. D.
Sweatt. 1988. Sequencing of proteins from two-dimensional gels by using in
situ digestion and transfer of peptides to polyvinylidene difluoride mem-
branes: application to proteins associated with sensitization in Aplysia. Proc.
Natl. Acad. Sci. USA 85:7008–7012.
19. Kojima, S., A. Yano, T. Sasazuki, and N. Ohta. 1984. Associations between
HLA and immune responses in individuals with chronic schistosomiasis
japonica. Trans. R. Soc. Trop. Med. Hyg. 78:325–329.
20. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 227:680–685.
21. Lukacs, N. W., and D. L. Boros. 1991. Splenic and granuloma T-lymphocyte
responses to fractionated soluble egg antigens of Schistosoma mansoni-in-
fected mice. Infect. Immun. 59:941–948.
22. Marquet, S., L. Abel, D. Hillaire, H. Dessein, J. Kalil, J. Feingold, J. Weis-
senbach, and A. J. Dessein. 1996. Genetic localization of a locus controlling
the intensity of infection by Schistosoma mansoni on chromosome 5q31-q33.
Nat. Genet. 14:181–184.
23. Mathew, R. C., and D. L. Boros. 1986. Anti-L3T4 antibody treatment sup-
presses hepatic granuloma formation and abrogates antigen-induced inter-
leukin-2 production in Schistosoma mansoni infection. Infect. Immun. 54:
820–826.
24. Matsudaira, P. 1987. Sequence from picomole quantities of proteins elec-
troblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. 262:
10035–10038.
25. Moser, D., M. J. Doenhoff, and M. Q. Klinkert. 1992. A stage-specific
calcium-binding protein expressed in eggs of Schistosoma mansoni. Mol.
Biochem. Parasitol. 51:229–238.
26. Nene, V., D. W. Dunne, K. S. Johnson, D. W. Taylor, and J. S. Cordingley.
1986. Sequence and expression of a major egg antigen from Schistosoma
mansoni. Homologies to heat shock proteins and alpha-crystallins. Mol.
Biochem. Parasitol. 21:179–188.
27. Pearce, E. J., S. L. James, S. Hieny, D. E. Lanar, and A. Sher. 1988.
Induction of protective immunity against Schistosoma mansoni by vaccina-
tion with schistosome paramyosin (Sm97), a nonsurface parasite antigen.
Proc. Natl. Acad. Sci. USA 85:5678–5682.
Editor: J. M. Mansfield
1735
28. Pearce, E. J., P. Caspar, J. M. Grzych, F. A. Lewis, and A. Sher. 1991.
Downregulation of Th1 cytokine production accompanies induction of Th2
responses by a parasitic helminth, Schistosoma mansoni. J. Exp. Med. 173:
159–166.
29. Rabelo, E. M. L., G. R. Franco, V. Azevedo, H. B. Pena, T. M. Santos, W. S. F.
Maria, N. A. Rodrigues, J. M. Ortega, and S. D. J. Pena. 1996. Analysis of
cDNA libraries from different developmental stages of Schistosoma mansoni
with the aim of producing expressed sequence Tags. GenBank DNA se-
quence a140576.
30. Reynolds, S. R., C. B. Shoemaker, and D. A. Harn. 1992. T and B cell epitope
mapping of SM23, an integral membrane protein of Schistosoma mansoni.
J. Immunol. 149:3995–4001.
31. Reynolds, S. R., C. E. Dahl, and D. A. Harn. 1994. T and B epitope deter-
mination and analysis of multiple antigenic peptides for the Schistosoma
mansoni experimental vaccine triose-phosphate isomerase. J. Immunol. 152:
193–200.
32. Schweitzer, A. N. 1992. Alternative patterns of MHC-restricted antibody
responsiveness following intraperitoneal immunization of inbred mice with
different preparations of an 86 kilodalton antigen of Schistosoma mansoni.
Parasite Immunol. 14:267–277.
33. Secor, W. E., H. del Corral, M. G. dos Reis, E. A. Ramos, A. E. Zimon, E. P.
Matos, E. A. Reis, T. M. do Carmo, K. Hirayama, R. A. David, J. R. David,
and D. A. Harn, Jr. 1996. Association of hepatosplenic schistosomiasis with
HLA-DQB1*0201. J. Infect. Dis. 174:1131–1135.
34. Smithers, S. R., and M. J. Doenhoff. 1982. Schistosomiasis, p. 527–607. In S.
Cohen and K. S. Warren (ed.), Immunology of parasitic diseases. Blackwell
Scientific, Oxford, England.
35. Soisson, L. M., C. P. Masterson, T. D. Tom, M. T. McNally, G. H. Lowell,
and M. Strand. 1992. Induction of protective immunity in mice using a
62-kDa recombinant fragment of a Schistosoma mansoni surface antigen.
J. Immunol. 149:3612–3620.
36. Stadecker, M. J., and H. J. Hernandez. 1998. The immune response and
immunopathology in infection with Schistosoma mansoni: a key role of major
egg antigen Sm-p40. Parasite Immunol. 20:217–221.
37. Sung, C. K., and M. H. Dresden. 1986. Cysteinyl proteinases of Schistosoma
mansoni eggs: purification and partial characterization. J. Parasitol. 72:891–
900.
38. Tendler, M., C. A. Brito, M. M. Vilar, N. Serra-Freire, C. M. Diogo, M. S.
Almeida, A. C. Delbem, J. F. Da Silva, W. Savino, R. C. Garratt, N. Katz, and
A. S. Simpson. 1996. A Schistosoma mansoni fatty acid-binding protein,
Sm14, is the potential basis of a dual-purpose anti-helminth vaccine. Proc.
Natl. Acad. Sci. USA 93:269–273.
39. Tielens, A. G., P. Van der Meer, J. M. van den Heuvel, and S. G. van den
Bergh. 1991. The enigmatic presence of all gluconeogenic enzymes in Schis-
tosoma mansoni adults. Parasitology 102:267–276.
40. Tielens, A. G., A. M. Horemans, R. Dunnewijk, P. van der Meer, and S. G.
van den Bergh. 1992. The facultative anaerobic energy metabolism of Schis-
tosoma mansoni sporocysts. Mol. Biochem. Parasitol. 56:49–57.
41. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of
proteins from polyacrylamide gels to nitrocellulose sheets: procedure and
some applications. Proc. Natl. Acad. Sci. USA 76:4350–4354.
42. Trzyna, W. C., and J. S. Cordingley. 1993. Schistosoma mansoni: genetic
non-response to p40, the major protein antigen of the egg, reveals a novel
mechanism enhancing IgM production during infection. Parasite Immunol.
15:601–611.
43. Waine, G. J., A. G. Ross, G. M. Williams, A. C. Sleigh, and D. P. McManus.
1998. HLA class II antigens are associated with resistance or susceptibility to
hepatosplenic disease in a Chinese population infected with Schistosoma
japonicum. Int. J. Parasitol. 28:537–542.
44. Watson, J. 1979. Continuous proliferation of murine antigen-specific helper
T lymphocytes in culture. J. Exp. Med. 150:1510–1519.
45. Wilkinson, J. M. 1986. Fragmentations of polypeptides by enzymatic meth-
ods, p. 122–148. In A. Darbre (ed.), Practical protein chemistry: a handbook.
John Wiley & Sons, New York, N.Y.
46. Wynn, T. A., I. Eltoum, A. W. Cheever, F. A. Lewis, W. C. Gause, and A. Sher.
1993. Analysis of cytokine mRNA expression during primary granuloma
formation induced by eggs of Schistosoma mansoni. J. Immunol. 151:1430–
1440.